title: Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery.
abstract: Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G(i/o) proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G(i/o) proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser(1177), which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr(14), which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G(i/o)-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser(1177) leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr(14), through a G(i/o)-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.
authors.affiliation: INSERM U1045, Centre de recherche cardiothoracique de Bordeaux, Bordeaux, France. sebastien.banquet@inserm.fr
authors.forename: Sébastien
authors.initials: S
authors.lastname: Banquet
authors.forename: Estelle
authors.initials: E
authors.lastname: Delannoy
authors.forename: Abdelali
authors.initials: A
authors.lastname: Agouni
authors.forename: Chantal
authors.initials: C
authors.lastname: Dessy
authors.forename: Sabrina
authors.initials: S
authors.lastname: Lacomme
authors.forename: Fabien
authors.initials: F
authors.lastname: Hubert
authors.forename: Vincent
authors.initials: V
authors.lastname: Richard
authors.forename: Bernard
authors.initials: B
authors.lastname: Muller
authors.forename: Véronique
authors.initials: V
authors.lastname: Leblais
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology; D000818:Animals; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D015237:Dinoprost / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D017353:Gene Deletion; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017265:Procaterol / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D051242:Caveolin 1; D004791:Enzyme Inhibitors; D018343:Receptors, Adrenergic, beta-2; D014665:Vasodilator Agents; D015237:Dinoprost; D052250:Nitric Oxide Synthase Type III; D037342:Pertussis Toxin; D017853:Phosphotransferases (Alcohol Group Acceptor); D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000109:Acetylcholine; D017265:Procaterol